Mutations in the ubiquitin E3 ligase component pVHL are observed in 100% of patients with the autosomal dominant von Hippel-Lindau disease (VHL) and predisposes individuals to a variety of tumors including clear cell carcinomas of the kidneys. Furthermore, mutations in pVHL are observed in 50-80% of patients with the more common sporadic renal clear cell carcinoma. Following the clinical success of the proteasome inhibitor bortezomib, ubiquitin pathway enzymes have been considered attractive targets to screen for inhibitors more selective than bortezomib thereby achieving an improved therapeutic index. Two related deubquitinating enzymes -- VDU1, now called USP33, and VDU2, now called USP20 -- are substrates of the pVHL E3 ligase complex and hence are implicated in carcinogenesis. The tight regulation of USP20 and USP33 protein levels by the tumor suppressor pVHL support the hypothesis that USP20 and USP33 proteins play a role in carcinogenesis. Approximately two years ago, in a phase I grant application it was proposed to establish an assay to screen USP33 (VDU1) isopeptidase activity using a reporter enzyme that required a free N terminus for catalytic activity. Subsequently, recombinant USP33 was purified and a high throughput assay for screening for inhibitors was established, fulfilling the main objective of the phase I grant (see phase I final report). In this phase II application the related deubiquitinase VDU2 (USP20) will be purified and the isopeptidase assay will be utilized to screen multiple libraries of compounds to identify inhibitors of USP33 and USP20. The hits will be confirmed by additional secondary assays to establish the specificity and efficacy of the inhibitors. Following lead optimization, initial preclinical evaluation will be performed. The identification of specific inhibitors of USP33 and/ or USP20 is the first step in the development of novel targeted therapies for pVHL mutant tumors. Such targeted therapies are predicted to be efficacious in the treatment of multiple malignancies such as renal clear cell carcinomas. In a Phase I SBIR grant funded project, Progenra, Inc developed an assay that can be used to find compounds that block an enzyme called VDU1 (or USP33), which is related to certain kidney cancers. In Phase II, it is proposed to use this assay to screen a large number of compounds to find the best candidates for a drug that can treat kidney cancer by blocking this enzyme or its relative, VDU2 (now called USP20). In the proposed work plan for Phase II, USP20 will be purified, and both USP20 and USP33 will be put into the screen to find compounds that are strong blockers of the enzymes (one or the other, or both). Finding powerful inhibitors of VDU1 or VDU2 is the first step along the pathway to develop new cancer drugs. [unreadable] [unreadable] [unreadable]